This revised application is a competing renewal of a proposal to investigate radiation-induced genomic instability. The goal of this application is to consolidate the "instability program" and focus on the molecular mechanisms and biological consequences of radiation-induced instability. The proposed studies are an expansion of current research from the PI's laboratory and will build upon observations made during the previous funding period. We propose to continue to validate a plasmid based recombination assay as a rapid and reliable indicator of delayed instability in cells post irradiation. Furthermore, we will utilize the extensive collection of chromosomally unstable clones of cells we have generated during the previous funding period to investigate the role of instability in radioresistance and determine differences in gene expression that may be responsible for the observed instability. To accomplish these goals there are three specific aims: Specific aim 1 will test the hypothesis that a recombination assay based on expression of green fluorescence protein can be used to rapidly and efficiently identify unstable clones as a function of time after irradiation. We will characterize this plasmid based assay system in relationship to our well defined chromosomal assay and use it to determine the effects of fractionated radiation treatment, DNA repair inhibitors, and modifying the post irradiation on induced instability. Specific aim 2 will expand upon our observation that genomic instability contributes to cellular radioresistance and we will investigate the hypothesis that this is due to differential gene expression. Specific aim 3 will test the hypothesis that radiation-induced genomic instability has a molecular/genetic basis reflecting differential gene expression, and that differential display strategies and/or RNA hybridization strategies using microarray technology can be used to identify the gene(s) involved in initiating and/or perpetuating the unstable phenotype.